共查询到20条相似文献,搜索用时 8 毫秒
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In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes 总被引:3,自引:0,他引:3
Ishii T Yasuchika K Fujii H Hoppo T Baba S Naito M Machimoto T Kamo N Suemori H Nakatsuji N Ikai I 《Experimental cell research》2005,309(1):68-77
It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes. 相似文献
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Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction
of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells
differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation
events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up
to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic
stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For
example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated
expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular
therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the
transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective
enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These
cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes.
However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating
that chondrocytes derived from ES cells posses a distinct differentiation plasticity.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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In vitro gametogenesis from embryonic stem cells 总被引:3,自引:0,他引:3
Many insights into mammalian germ cell development have been gained through genetic engineering and in vivo studies, but the lack of an in vitro system for deriving germ cells has hindered potential advances in germ cell biology. Recent studies have demonstrated embryonic stem cell differentiation into germ cells and more mature gametes, although significant unanswered questions remain about the functionality of these cells. The derivation of germ cells from embryonic stem cells in vitro provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitate nuclear transfer technology and infertility treatments. 相似文献
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Pellett S Du ZW Pier CL Tepp WH Zhang SC Johnson EA 《Biochemical and biophysical research communications》2011,(1):388-392
Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA. 相似文献
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Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells 总被引:4,自引:0,他引:4
Tian HB Wang H Sha HY Xu XJ Zhu M Wu YB Cheng SH Chen JQ Shi YX Bai ZL Cheng GX 《Cell biology international》2006,30(5):452-458
Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells. 相似文献
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Nakano Y Hirko AC Smith AD Oka M Dawson R Peris J Terada N Meyer EM 《Neurochemistry international》2004,45(7):1067-1073
This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter. 相似文献
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Labelling of mammalian cells with superparamagnetic iron oxide (SPIO) nanoparticles enables to monitor their fate in vivo using magnetic resonance imaging (MRI). However, the question remains whether or not SPIO nanoparticles affect the phenotype of labelled cells. In the present study, the effects of SPIO nanoparticles from two producers on the growth and differentiation of mouse embryonic stem (ES) cells in vitro were investigated. Our observations have shown that SPIO nanoparticles have no effect on the self-renewal of ES cells. Subsequently, we studied the effect of SPIO on the formation of embryoid bodies and neural differentiation of ES cell in monolayer culture. The cavitation of embryoid bodies was partially inhibited and neural differentiation was supported regardless the type of SPIO nanoparticles used. Thus for the first time we documented the effects of SPIO nanoparticles on ES cells and their differentiation. 相似文献
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Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases. 相似文献
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Jen-Hua Chuang Li-Chu Tung Guey-Jen Lee-Chen Yi Yin Yenshou Lin 《Analytical biochemistry》2011,(1):149
Much effort is being marshaled to generate uniform neuronal populations from embryonic stem (ES) cells, but a completely reliable method has yet to be developed. Here we modified and established a method that brings us closer to this goal. By examining many parameters, we found that the optimal timing of applying a freshly made trypsin/EDTA (ethylenediaminetetraacetic acid) solution to dissociate embryoid bodies determines the success of the outcome. Analyses demonstrated that with this approach, more than 87% of cells differentiated into glutamatergic neurons. Hence, these uniform neurons that were differentiated from ES cells provide an ideal cellular model for many aspects of research. 相似文献
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The evaluation of antibody avidity by elution with chaotropic agents is a frequently used approach in research and diagnostics. It provides important information on the functional relevance of antibodies. However, in the literature, there is a large heterogeneity in the experimental settings for the determination of this important parameter. Here, we demonstrate that antibody concentration and the choice of chaotropic agent are critical for the reliable estimation of antibody avidity. 相似文献
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Qiuyue Liu Oliver Z. Pedersen Jun Peng Larry A. Couture Mahendra S. Rao Xianmin Zeng 《Cytotherapy》2013,15(8):999-1010
Background aimsWe have previously described a xeno-free scalable system to generate transplantable dopaminergic neurons from human pluripotent stem cells. However, several important questions remain to be answered about our cell therapy efforts. These include determining the exact time at which cells should be transplanted and whether cells at this stage can be frozen, shipped, thawed and injected without compromising their ability to mature and survive the transplantation procedure. We also needed to determine whether further optimization of the culture process could shorten the development time and reduce variability and whether a current Good Manufacture Practice (CGMP) facility could manufacture cells with fidelity.MethodsWe developed an optimized protocol that included modulating the sonic hedgehog homolog gradient with bone morphogenetic proteins (BMP2) and addition of activin to the culture medium, which shortened the time to generate Lmx1A and FoxA2 immunoreactive cells by 4–6 days.ResultsWe showed that cells at this stage could be safely frozen and thawed while retaining an excellent ability to continue to mature in vitro and survive transplant in vivo. Importantly, we successfully adapted this process to a CGMP facility and manufactured two lots of transplant-ready dopaminergic neurons (>250 vials) under CGMP-compatible conditions. In vitro characterization, including viability/recovery on thawing, whole genome expression as well as expression of midbrain/dopaminergic markers, showed that the cells manufactured under GMP-compatible conditions were similar to cells produced at lab scale.ConclusionsOur results suggest that this optimized protocol can be used to generate dopaminergic neurons for Investigational New Drug enabling studies. 相似文献
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The demonstration of germ cell and haploid gamete development from embryonic stem cells (ESCs) in vitro has engendered a unique set of possibilities for the study of germ cell development and the associated epigenetic phenomenon. The process of embryoid body (EB) differentiation, like teratoma formation, signifies a spontaneous differentiation of ESCs into cells of all three germ layers, and it is from these differentiating aggregates of cells that putative primordial germ cells (PGCs) and more mature gametes can be identified and isolated. The differentiation system presented here requires the differentiation of murine ESCs into EBs and the subsequent isolation of PGCs as well as haploid male gametes from EBs at various stages of differentiation. It serves as a platform for studying the poorly understood process of germ cell allocation, imprint erasure and gamete formation, with 4-6 weeks being required to isolate PGCs as well as haploid cells. 相似文献
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正Embryonic stem cells(ESCs)are pluripotent and can self-assemble to form cell clusters or embryoid bodies(EBs),which can then differentiate into all cell types of the three germ layers,as among which one is the primordial germ cell(PGCs)(Daley,2007).In vivo PGCs are the gamete founder cells(the ooctyes and sperms),which transmit genetic information from one generation to the next generation to maintain mammalian life cycles.About 15%of couples in China are infertile;some of these infertility cases are 相似文献
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Akama K Tatsuno R Otsu M Horikoshi T Nakayama T Nakamura M Toda T Inoue N 《Biochimica et biophysica acta》2008,1784(5):773-782
Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons. 相似文献
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I. V. Kozhukharova I. I. Fridlyanskaya V. I. Zemel’ko Z. V. Kovaleva N. A. Pugovkina L. L. Alekseenko M. V. Charchenko N. D. Aksenov A. N. Shatrova T. M. Grinchuk S. V. Anisimov N. N. Nikol’skii 《Cell and Tissue Biology》2010,4(5):411-418
The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESCs) in vitro. It was shown that human ESCs can be differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by the successive application of noggin and bFGF growth factors and collagen and matrigel substrates for 3–4 weeks. The efficiency of differentiation was evaluated by the number of colonies with cells that express tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension estimated by flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. The lack of pluripotent cells in population at the final stage of differentiation is encouraging and shows that this protocol of human ESC differentiation may be applied to generate DA neurons for their transplantation into the animals modeling neurodegenative (Parkinson) disease without the risk of tumor growth. 相似文献
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Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells 总被引:18,自引:0,他引:18
Hancock CR Wetherington JP Lambert NA Condie BG 《Biochemical and biophysical research communications》2000,271(2):418-421
Embryonic stem cells (ES cells) are developmentally pluripotent cells isolated from pre-implantation mammalian embryos. In cell culture ES cells can be easily differentiated to generate cultures of neural progenitors. We present a simple method for the cryopreservation of these ES-derived neural progenitors. Cryopreserved neural progenitor stocks can be thawed, expanded with FGF2, and differentiated into functional neurons. This method will facilitate studies using ES-derived neural progenitor cells as a cell culture model system for neural development and differentiation. It will also aid studies designed to test the ability of these progenitor cells to functionally engraft and repair damaged neural tissue. 相似文献